Project Summary (Abstract) Treatment of patients with anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC) has been greatly improved over the past decade by the use of small molecule kinase inhibitors to target ALK. Although these treatments have drastic initial responses, patients on average develop resistance to these inhibitors within a year of treatment. All current ALK targeted therapies, both approved and in clinical trials, are ATP-competitive kinase inhibitors. A new class of compounds that target ALK may provide ways of overcoming tumor resistance, either on its own or through combination therapies with ATP-competitive inhibitors. The goal of the proposed research is to identify and validate compounds that target ALK-positive NSCLC through a novel mechanism of action, and demonstrate their ability to overcome resistance mutations. This will be accomplished through two main aims: the identification and profiling of allosteric ALK inhibitors (Aim 1) and the optimization and profiling of ALK degrader compounds (Aim 2). Based on the hypothesis that ALK contains an allosteric site similar to that of the epidermal growth factor receptor (EGFR), allosteric ALK inhibitors will be identified by a high-throughput compound screen, before being profiled both biochemically and in cells. Co- crystal structures of the allosteric inhibitors with ALK will provide critical information to guide medicinal chemistry efforts to improve the potency and selectivity of these inhibitors. Lead ALK degrader compounds have already been synthesized by our group based on recently reported studies that demonstrated the use of hetero-bifunctional small molecules to induce the degradation of protein targets. These degrader compounds are composed of an inhibitor to engage the target protein and a phthalimide immunomodulatory drug that recruits the cereblon (CRBN) E3 ubiquitin ligase to ubiquitinate the target protein, leading to proteasomal degradation. Biochemical assays will be used to demonstrate target engagement and successful formation of the ternary complex between ALK, CRBN, and the degrader compounds. Cellular assays will be used to validate the ability of the bifunctional small molecules to induce ALK degradation and explore the degraders' anti-proliferative effects and effects on downstream signaling. These biochemical and cellular assays will inform medicinal chemistry efforts to optimize the pharmacological properties of the degrader compounds. Successful completion of either of these two aims will provide proof of concept that a new class of compounds may be used to therapeutically target ALK-positive NSCLC.